We will develop and apply new biophysical approaches to the structure determination of large nucleic acids and nucleoprotein assemblies. Psoralen derivatives will be adapted into bis-double strand crosslinkers, protein-nucleic acid crosslinkers, and a variety of photo affinity label analogs. Gel electrophoretic and electron microscopic approaches for identification of crosslinking sites will be refined. These techniques will be used to work out the detailed packaging arrangement of DNA in bacteriophage lambda. They will also be used to examine the mechanism of refolding of complex single strand nucleic acids such as 16S rRNA. Of particular interest is how such molecules avoid or attain knotted topologies. We will exploit unusual nucleotides to direct the incorporation of crosslinkers or fluorescent probes to specific places in nucleic acid sequences. Pilot studies will examine the utility of such approaches in studies of replication and the E. coli nucleoid. We will also study the chemical details of repair of double strand crosslinks in E. coli by infection with bacteriophage lambda carrying crosslinks at precise sequence locations. Such studies could well provide important details about the mechanism of recombination.